Transient Host–Guest Complexation To Control Catalytic Activity

Signal transduction mechanisms are key to living systems. Cells respond to signals by changing catalytic activity of enzymes. This signal responsive catalysis is crucial in the regulation of (bio)chemical reaction networks (CRNs). Inspired by these networks, we report an artificial signal responsive system that shows signal-induced temporary catalyst activation. We use an unstable signal to temporarily activate an out of equilibrium CRN, generating transient host–guest complexes to control catalytic activity. Esters with favorable binding toward the cucurbit[7]uril (CB[7]) supramolecular host are used as temporary signals to form a transient complex with CB[7], replacing a CB[7]-bound guest. The esters are hydrolytically unstable, generating acids and alcohols, which do not bind to CB[7], leading to guest reuptake. We demonstrate the feasibility of the concept using signal-controlled temporary dye release and reuptake. The same signal controlled system was then used to tune the reaction rate of aniline catalyzed hydrazone formation. Varying the ester structure and concentration gave access to different catalyst liberation times and free catalyst concentration, regulating the overall reaction rate. With temporary signal controlled transient complex formation we can tune the kinetics of a second chemical reaction, in which the signal does not participate. This system shows promise for building more complex nonbiological networks, to ultimately arrive at signal transduction in organic materials.


General materials and methods
Chemicals were purchased in the highest purity and used without further purification unless reported otherwise. Tetrahydrofuran (THF), methanol and diethyl ether (DE) of technical grades were purchased from VWR International. Acridine orange 5 (AO) hydrochloride solution (10 mg/mL) and aniline catalyst 6 (99.5%) of ACS reagent grade were purchased from Sigma Aldrich. CB [7] hydrate was purchased from Strem Chemicals Inc, and based on ITC measurements it was estimated to contain about 30 wt% hydration water. o-Sulfobenzaldehyde 7 was purchased from Honeywell Fluka Fischer Scientific and p-hydroxybutiric acid hydrazide 8 (98%) from Alfa Aesar. Methyl bromoacetate (99%), ethyl bromoacetate (98%), isopropyl bromoacetate (99%) and trimethylamine solution (31-35 wt% ethanol) were purchased from Sigma Aldrich. Solid salts were used for the preparation of aqueous buffers. Sodium phosphate monobasic monohydrate (98%) was purchased from Sigma Aldrich, while sodium phosphate dibasic (+99%, analysis grade) was from Acros Organics. Unless stated otherwise, all preparations and analyses were performed at room temperature (RT) (~21 ○ C) and atmospheric pressure. Nuclear Magnetic Resonance (NMR) experiments were performed using Agilent-400 MR DD2 (400 MHz for 1 H and 100.5 MHz for 13 C) at 25 ○ C using residual deuterated solvent signals as internal standard. To suppress the water peak, PRESAT configuration (suppress one highest peak) was used. UV-Vis spectroscopic experiments were carried out using Analytik Jena Specord 250 spectrophotometer; quartz cuvette with a 1 cm path length, volume of 3 mL, at a controlled temperature of 25 ○ C. Fluorescence spectra were recorded on a JASCO J-815 CD spectrometer (sensitivity 450 Volts, data pitch 1 nm, band width 5 nm, excitation wavelength of 465 nm for AO 5); black quartz cuvette with a 1 cm path length, volume of 200 µL, at RT. Isothermal titration calorimetry (ITC) experiments were performed using a MicroCal VP-ITC. Liquid Chromatography-Mass Spectrometry (LC-MS) was performed on a Shimadzu Liquid Chromatograph Mass Spectrometer 2010, LC-8A pump with a diode array detector SPD-M20. Negative and/ or positive mode Electro Spray Ionization Mass Spectrometry (ESI-MS) was used for the peak assignment.

Glycine betaine esters (methyl 1, ethyl 2 and isopropyl 3) synthesis
Following the literature [1], for methyl ester 1, trimethylamine (1.5 equivalents; 33 wt % in EtOH) was added to a solution of methyl bromoacetate (1 equivalent) in THF (50 mL/g). The reaction mixture was stirred for 24h at RT, during this time the product precipitated as a white solid. The resulting suspension was cooled with ice, filtered, washed with ice-cold Et2O and dried in the vacuum oven at 40 ○ C overnight to yield the ammonium salt with bromine counter ion as white solid. For the ethyl and isopropyl esters (2-3) the same procedure was followed with ethyl bromoacetate or isopropyl bromoacetate respectively.

Hydrazone product 9 synthesis
The synthesis was performed according to our previous reported procedure [2]. The purity of the product was confirmed by NMR and MS: N.B. Extra splitting of the peaks in the NMR spectrum is due to cis and trans isomers. 1

ITC binding constant measurement
A solution of the guest molecule (3x10 -5 mol from 10 mM stock) was titrated to CB[7] (0.35 mM) solution at 25 ○ C. Unless stated differently, the solutions were prepared in sodium phosphate buffer 100 mM pH 7.5. The first titration point out of 28 injection points was discarded. Binding constants were fitted with Microcal LLC ITC Origin 7 software.

Fluorescence assay of acridine orange 5 and CB[7]
Fluorescence measurements were performed in 100 mM sodium phosphate buffer pH 7.5 with 0.027 mM AO 5, 0.054 mM CB[7] and esters (2.68 mM methyl 1, 0.67 mM ethyl 2 and 0.13 mM isopropyl 3) in black quartz cuvettes, path length of 1 cm (total reaction volume of 200 µL) at RT. Based on these concentrations, 96% of the CB[7] will be occupied by methyl ester 1, and 96% by ethyl ester 2 and 97% by isopropyl ester 3. The stock solutions were added in the following order: phosphate buffer, dye 5 and CB[7] (premixed for 1 h) and ester 1-3 solution. Teflon caps were used to close the cuvette. The cuvette was turned upside down to mix the solution. Samples were excited at wavelength 465 nm. N.B., AO 5 has a pKa of 9.8 and is predominantly present in the protonated form at pH 7.5 [3].

UV-Vis assay to follow the hydrazone formation reaction
Unless stated otherwise, the hydrazone reaction was performed in 100 mM sodium phosphate buffer pH 7.5, containing 0.2 mM aldehyde 7, 0.02 mM hydrazide 8, 0.2 mM aniline 6, and 0.6 mM CB[7] in quartz cuvettes, path length of 1 cm (total reaction volume of 3 mL) at 25 ○ C. The stock solutions of the reactants were added in the following order: catalyst 6 solution and CB[7] (premixed for 1 h), aldehyde 7 solution, phosphate buffer and hydrazide 8 solution. Teflon caps were used to close the cuvette. The cuvette was turned upside down five times to mix the solution. The product peak was followed (at 287 nm) using a 6-sample holder (configuration: slow time scan, scan every 20 s). The pH was measured before and after the reaction. Experiments with esters 1-3 (2.5/ 2 mM methyl 1, 1.5/ 1 mM ethyl 2 and 0.8/ 0.75 mM isopropyl 3) were performed similarly, with the ester 1-3 stock solution being added at the last moment. Based on these concentrations, 95%/ 94% of the methyl ester 1 will bind to CB[7], 97%/ 95% of ethyl ester 2 and 99%/ 99% of isopropyl ester 3. The concentration of hydrazone product 9 was calculated with the extinction coefficient and Lambert-Beer law.   Table S1: Binding constants (Ka) and thermodynamic binding values (ΔH and TΔS) for esters 1-3, their hydrolysis products, acridine orange (AO) 5, aniline catalyst 6, aldehyde 7, hydrazide 8 and hydrazine 9 as determined by Isothermal titration calorimetry (ITC) or from the literature. A negative ΔH value indicates an enthalpic gain, while a positive TΔS indicates an entropic gain. Not measurable means that the binding constant cannot be measured, because it is under the detection limit of the ITC. 1 The binding constant for AO 5 was taken from the literature obtained with fluorescence titration.         Using Lambert-Beer law: = , where is the absorbance of 9 at 287 nm, is the extinction coefficient, is the path length and is the concentration of hydrazone 9, is the slope of plot in Figure S12B. Therefore, extinction coefficients for hydrazone 9 at 287 nm is 19.2 mM -1 cm -1 at pH 7.5.

Ester hydrolysis profiles
The yield of hydrazone product 9 is further calculated by determining the concentration from: By monitoring the absorbance of product 9 peak at 287 nm, the yield of 9 as a function of time can be calculated. All graphs showing the yield of 9 in this work are calculated in this way.

Kinetic model
In this section the kinetic model is explained, which calculates the changing reaction rate constant for hydrazone formation during the cycle. When the esters are added to the reaction mixture, the catalyst is liberated from the CB[7] host and over time due to the hydrolysis of the esters the catalyst is captured again inside CB [7]. Consequently, the free catalyst concentration in the solution is changing as a function of time (and the catalytic rate constant). Overall, this can be modelled numerically with a set of differential equations. To do this, we will first explain the ester hydrolysis kinetics, hydrazone formation kinetics and equilibrium concentration calculations for host-guest complex formation. After that we assemble the kinetic model based on these individual parts and numerically calculate the concentration of the different species and the k-value.

Ester hydrolysis reaction kinetics
It is assumed that ester hydrolysis proceeds in a pseudo-first order rate: where !"#$%&"'(' is the hydrolysis rate constant and [ ] the time-dependent ester concentration. After integration, the rate law can be written as: where [ ] * is the initial ester concentration, [ ] ) the ester concentration at time t and !"#$%&"'(' the hydrolysis rate constant. By following the ester vs acid concentration over time with 1 H NMR the hydrolysis rate constant can be determined by fitting the equation to a linear line (y = mx). The as such determined rate constants for ester hydrolysis at different pH and inside or outside CB[7] are provided in Figure S6-7.

Hydrazone formation reaction kinetics
The hydrazone formation reaction is assumed to be a second-order reaction. The reaction was performed at pseudo-first order conditions by using one of the reagents in excess. Unless stated otherwise, concentrations used in the hydrazone formation reaction were 0.2 mM aldehyde 7, 0.02 mM hydrazide 8.

Matlab numerical model of differential equations
Using the above relations and by determining the ester hydrolysis rate constants in and outside CB[7] with 1 H NMR, the catalyst in the solution as a function of time can be calculated and based on that the rate constant for hydrazone formation over time. Upon addition of the esters, the esters will form a complex with CB[7] with a concentration of L ÌCB[7]O( ), which is a function of time (as the esters hydrolyse over time). The ester inside CB[7] hydrolyses slower than outside CB[7] this is taken into account in the model. Hence, to calculate the concentration profiles of all the species in the reaction mixture, we use the following equations and use the Matlab function ode45 to calculate them numerically:

NMR and MS controls
A long-term 1 H NMR measurement was performed for the aniline catalysed hydrazone formation reaction in the presence of esters 1-3 to verify the formation of any possible side products (Conditions: 2 mM aldehyde 7, 0.2 mM hydrazide 8 and 2 mM aniline 6, with methyl ester 1 25 mM, ethyl ester 2 15 mM, isopropyl ester 3, in pH 7.5, 100 mM sodium phosphate buffer solution and 10% D2O). However, due to the large difference in concentrations of reactants, catalysts and esters, the peaks cannot be visualized in one scale. Therefore, two scales of the same spectrum have been provided in Figure S21-S23 to show each peak.
On top of that, LC-MS of these reaction solutions after 8 days were also measured, however no identifiable side products were observed.